Alginate-based nanofibers and related scaffolds

ABSTRACT

Alginate nanofibers, scaffolds that include alginate nanofibers, implantable devices that include alginate nanofibers, and methods for making the alginate nanofibers by electrospinning.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Application No.PCT/US2007/065388, filed Mar. 28, 2007, which claims the benefit of U.S.Provisional Application No. 60/787,099, filed Mar. 28, 2006. Eachapplication is expressly incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

The invention was made with government support under Contract No. EEC9529161, awarded by the National Science Foundation. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Polymeric nanofibers can be used for a broad spectrum of biological andmedical applications. They are of particular interest in regenerativemedicine and tissue engineering because they can be potentially tailoredto mimic the natural extracellular matrix (ECM) in terms of structure,chemical composition, and mechanical properties. In this context, theyserve as scaffolds to direct cellular behavior and function until hostcells can repopulate and resynthesize a new natural matrix. The ECMmolecular network surrounding the cells provides mechanical support andregulates cellular activities. The natural ECM in human tissue is mainlycomposed of proteoglycans (glycosaminoglycan (GAG)) and fibrousproteins, both with nanoscale structural dimensions. Studies have shownthat scaffolds with nanoscale structures support cell adhesion andproliferation, and function better than their microscale counterparts.

A number of synthetic polymer nanofibers with fiber diameters from a fewtens to a few hundreds of nanometers have been fabricated for tissueengineering; these include polyglycolide (PGA), poly(L-lactic acid)(PLA), and their copolymers poly(glycolide-co-lactide) (PLGA) andpoly(ε-caprolactone) (PCL). The studies demonstrated favorablebiological responses of seeded cells, such as enhanced cell attachmentand in vitro proliferation. Recently, there has been growing interest inthe synthesis of natural polymer-based nanofibers because of theirproven biocompatibility and their resorbable biodegradation products.Advantageous attributes of natural polymers include hydrophilicity,non-toxicity, less-immune reaction, as well as enhanced cell adhesionand proliferation. Collagen, gelatin, hyaluronan, chitosan, and alginateare the most commonly used natural polymers in tissue engineering. In afew recent studies, collagen and chitosan have been successfullyfabricated into nanofibers and have demonstrated good cellularcompatibility. The ability to generate nanofibrous matrices from naturalpolymers, especially those derived from plants, may provide virtuallyunlimited resources for the development of tissue-compatible scaffoldsfor functional restoration of damaged or dysfunctional tissues. Thisrestoration currently relies mainly on the autograft and allograftprocedures—surgical procedures facing the challenges of limitedresources, risk of infection, and viral transmission.

SUMMARY OF THE INVENTION

The present invention provides alginate-based nanofibers, scaffolds thatinclude nanofibers, implantable devices that include the nanofibers, andmethods for making the nanofibers by electrospinning.

In one aspect, the invention provides an alginate-based fiber. In oneembodiment, the fiber includes alginate and has a diameter of from about20 to about 2000 nm. In one embodiment, the fiber has a diameter fromabout 50 to about 500 nm. In addition to alginate, the alginate-basedfiber of the invention includes a hydrophilic polymer to facilitateproduction of the fiber by electrospinning.

In one embodiment, the fiber includes from about 30 to about 95 weightpercent alginate based on the total weight of the fiber. In oneembodiment, about 80 weight percent alginate based on the total weightof the fiber.

Hydrophilic polymers useful in making the fiber of the invention includepoly(alkylene oxide) polymers, polyvinyl alcohol polymers, andpolycarboxylic acid polymers. Representative poly(alkylene oxide)polymers include polyethylene oxide polymers and block copolymers (forexample, nonionic block copolymers commercially available under thedesignation PLURONIC and/or POLOXAMER). In one embodiment, thehydrophilic polymer (e.g., poly(alkylene oxide)) has a molecular weightof from about 50 kDa to about 1000 kDa. In one embodiment, the fiberincludes from about 5 to about 30 weight percent poly(alkylene oxide)based on the total weight of the fiber. In one embodiment, the fiberincludes about 20 weight percent poly(alkylene oxide) based on the totalweight of the fiber.

The fiber of the invention can be crosslinked to maintains itsstructural integrity in aqueous and biological environments.

In one embodiment, the fiber of the invention is crosslinked with anionic crosslinking agent. Suitable ionic crosslinking agents include ametal ions reactive toward alginate carboxylic acid groups.Representative metal ions include calcium (2), barium (2), strontium(2), copper (2), zinc (2), magnesium (2), manganese (2), cobalt (2),lead (2), iron (3), and aluminum (3) ions.

In one embodiment, the fiber of the invention is crosslinked with acovalent crosslinking agent. Suitable covalent crosslinking agentsinclude bifunctional crosslinking agents reactive toward alginatehydroxyl and/or carboxylic acid groups. Representative covalentcrosslinking agents include carbodiimides, allyl halide oxides,dialdehydes, diamines, and diisocyanates. In certain embodiments, thecovalent crosslinking agent is selected from epichlorohydrin,gluteraldehyde, hexamethylene diisocyanate, adipic acid hydrazide, and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

In one embodiment, the fiber of the invention is both ionicallycrosslinked and covalently crosslinked.

In one embodiment, the fiber of the invention has a Young's modulus offrom about 0.004 to about 40 MPa.

In one embodiment, the fiber includes from about 30 to about 95 weightpercent alginate and from about 5 to about 70 weight percentpolyethylene oxide. In one embodiment, the fiber includes about 80weight percent alginate and about 20 weight percent polyethylene oxide.

In another aspect of the invention, fibrous scaffolds are provided. Thefibrous scaffolds of the invention include a plurality of the fibers ofthe invention, as described above.

In one embodiment, the scaffold having a thickness of from about 0.05 toabout 5 mm.

The scaffolds can include one or more materials other than the fibers ofthe invention.

In one embodiment, the scaffold further includes chitosan. In thisembodiment, the scaffold includes from about 0.5 to about 15 weightpercent chitosan.

In one embodiment, the scaffold further includes collagen. In thisembodiment, the scaffold includes from about 0.5 to about 20 weightpercent chitosan.

In one embodiment, the scaffold further includes a biologically activeamino acid molecule. Representative amino acid molecules include growthfactors, fibronectin, laminin, vitronectin, integrins, nucleic acidmolecules, lipids, sugars, antisense molecules, anti-inflammatoryagents, immunomodulatory agents, molecules that promote cell migration,molecules that promote cell division, molecules that promote cellproliferation and differentiation, molecules that stimulate phenotypicmodification of cells, molecules that promote angiogenesis, moleculesthat promote vascularization, and molecules that promote extracellularmatrix disposition.

In one embodiment, the scaffold further includes a signaling ligand.Representative signaling ligands include members of the TGF-β family,bone morphogenic proteins, fibroblast growth factors-1 and -2,platelet-derived growth factor-AA and -BB, and platelet rich plasma andvascular endothelial cell-derived growth factor. In this embodiment, thescaffold includes from about 1 to about 100 ng signaling ligand per gramscaffold.

In a further aspect of the invention, implantable devices are provided.The implantable devices include the fibers and/or the scaffolds of theinvention described above. Representative implantable devices includecell transplantation devices, drug delivery devices, wound dressings andhemostats, surgically implantable devices for repairing damagedcartilage in mammalian articulating joints, fabric barriers tohyperplasia and tissue adhesion, vascular prostheses, nerve grafts,spinal fusion cages, and skin substitutes.

In another aspect, the invention provides a method for making thealginate fibers of the invention. In one embodiment, the method includes

(a) generating an electrostatic field between a first electrode and asecond electrode; and

(b) electrospinning an aqueous alginate solution comprising alginate anda hydrophilic polymer onto a collection surface located between thefirst electrode and the second electrode to provide a plurality ofalginate fibers on the collection surface.

In one embodiment, the alginate solution includes from about 1 to about8 percent by weight alginate. In one embodiment, the alginate solutionincludes from about 3 to about 5 percent by weight alginate.

In one embodiment, the hydrophilic polymer is selected from apoly(alkylene oxide), a polyvinyl alcohol, and a polycarboxylic acidpolymer. In one embodiment, the alginate solution includes from about 1to about 10 percent by weight poly(alkylene oxide). In one embodiment,the alginate solution includes from about 2 to about 4 percent by weightpoly(alkylene oxide). In one embodiment, the poly(alkylene oxide) ispolyethylene oxide.

In one embodiment, the ratio of alginate to poly(alkylene oxide) is fromabout 70:30 to about 95:5. In one embodiment, the ratio of alginate topoly(alkylene oxide) is about 80:20.

In one embodiment, the alginate solution further includes a co-solvent.Suitable co-solvents are polar aprotic solvents. Representativeco-solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile,dimethylformamide, and dimethylsulfoxide. In one embodiment, theco-solvent is dimethylsulfoxide. In one embodiment, the co-solvent ispresent in the solution in an amount from about 0.5 to about 20 weightpercent. In one embodiment, the co-solvent is present in the solution inan amount from about 1 to about 10 weight percent. In one embodiment,the co-solvent is present in the solution in about 5 weight percent.

In one embodiment, the alginate solution further includes a surfactant.Suitable surfactants include glycol stearate, cocoamide surfactants, andpolyoxyethylene(10) isooctylphenyl ether (Triton X-100 family). In oneembodiment, the surfactant is present in the solution in an amount fromabout 0.01 to about 1.0 weight percent. In one embodiment, thesurfactant is present in the solution in about 0.05 weight percent. Inone embodiment, the surfactant is polyoxyethylene(10) isooctylphenylether.

In one embodiment, the alginate solution includes both a co-solvent anda surfactant, as described above.

In one embodiment, the zero shear viscosity of the alginate solution isfrom about 2.0 to about 8.0 Pa·s.

In one embodiment, the method further includes treating the plurality ofalginate fibers with an ionic crosslinking agent to provide a pluralityof ionically crosslinked alginate fibers. Suitable ionic crosslinkingagents are described above and include metal ions, such as calcium (2)ion, barium (2) ion, strontium (2), copper (2), zinc (2), magnesium (2),manganese (2), cobalt (2), lead (2), iron (3), and aluminum (3) ions.

In one embodiment, the method further includes treating the plurality ofionically crosslinked alginate fibers with a covalent crosslinking agentto provide a plurality of covalently crosslinked alginate fibers.Suitable covalent crosslinking agents are described above and includecarbodiimides, allyl halide oxides, dialdehydes, diamines, anddiisocyanates.

The fibers of the invention can be crosslinked ionically, covalently, orionically and covalently. In one embodiment, the method of the inventionincludes treating the alginate fibers with a covalent crosslinking agentto provide a plurality of covalently crosslinked alginate fibers.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIGS. 1A-1C′ are scanning electron microscope (SEM) images ofrepresentative alginate-polyethylene oxide (PEO) fibers of the inventionelectrospun from alginate/PEO solutions containing 0.5 wt % Triton X-100surfactant and 5 wt % DMSO co-solvent: FIG. 1A is an image of arepresentative alginate-PEO fiber spun from a solution having analginate/PEO ratio of 70:30; FIG. 1A′ is a higher magnification image ofthe image of FIG. 1A; FIG. 1B is an image of a representativealginate-PEO fiber spun from a solution having an alginate/PEO ratio of80:20; FIG. 1B′ is a higher magnification image of the image of FIG. 1B;FIG. 1C is an image of a representative alginate-PEO fiber spun from asolution having an alginate/PEO ratio of 90:10; and FIG. 1A′ is a highermagnification image of the image of FIG. 1A.

FIG. 2A is a bar graph comparing the tensile moduli of representativealginate-PEO fibers of the invention electrospun from an 80:20alginate/PEO solution: as spun fiber; calcium chloride crosslinked driedfiber; and calcium chloride crosslinked wet fiber.

FIGS. 2B and 2C are scanning electron microscope (SEM) images ofrepresentative alginate-polyethylene oxide (PEO) fibers of the inventionelectrospun from an 80:20 alginate/PEO solution after immersion indeionized (DI) water for 1 day and 15 days, respectively.

FIGS. 3A-3C compare the in vitro cellular compatibility of alginatefibers electrospun from an 80:20 alginate/PEO solution: FIG. 3A is ascanning electron microscope image of chondrocytes grown on alginate-PEOfibers; FIG. 3B is a higher magnification image of the image of FIG. 3B;and FIG. 3C is a fluorescence image of cells on fibers with theLive/Dead cell stain.

FIG. 4 is a graph comparing shear viscosities of polymer solutionshaving different alginate/PEO weight ratios (100:0; 90:10; 80:20; and60:40) as a function of shear rate.

FIGS. 5A-5D are SEM images of electrospun structures from alginatesolutions having alginate/PEO ratios of 90:10; 80:20; 70:30; and 60:40,respectively.

FIGS. 6A-6D are images of electrospun alginate/PEO fibers from 80:20alginate/PEO solutions: FIG. 6A is a SEM image of fibers obtained byadding Triton X-100 (0.5 wt %); FIG. 6B is a SEM image of fibersobtained by adding Triton X-100 (0.5 wt %) and DMSO (5.0 wt %); FIG. 6Cis a transmission electron microscope (TEM) image of the fibers of FIG.6B; and FIG. 6D is a higher magnification TEM image of the fibers ofFIG. 6B.

FIG. 7A is a graph illustrating zero shear viscosity of a 4 wt %alginate solution as a function of storage time.

FIG. 7B compares the infrared (FTIR) spectra of the 4 wt % alginatesolution as a function of storage time (Day 6, 10, 15, 20, and 30) tothe infrared spectrum of as-received alginate powder (Control).

FIG. 7C compares the ultraviolet (UV) absorbance spectra of the 4 wt %alginate solution as a function of storage time (Day 6, 10, 15, 20, and30) to the absorbance spectrum of as-received alginate powder (Control).

FIGS. 7D-7F are SEM images of fibers electrospun from alginate solutionshaving viscosities in Regions I, II, and III, respectively, of FIG. 7A.

FIGS. 8A and 8B are SEM images of a representative ionically (calciumchloride) crosslinked alginate (alginate/PEO 80:20) fibrous mat of theinvention incubated in DI water (FIG. 8A) and SBF (pH 7.4) (FIG. 8B) forseven days at 37° C.

FIGS. 8C-8F are SEM images of representative crosslinked alginate(alginate/PEO 80:20) fibrous mats of the invention incubated insimulated body fluid (SBF) (pH 7.4) for seven days at 37° C.: thefibrous mats were first ionically crosslinked with calcium chloridefollowed by covalent crosslinking with epichlorohydrin (FIG. 8C),glutaraldehyde (FIG. 8D), hexamethylene diisocyanate (HMDI) (FIG. 8E),or adipic acid hydrazide (ADA) (FIG. 8F).

FIG. 9A is a schematic illustration of a process of ionic and covalentcrosslinking and depicts the resultant associations between polymerchains in a single nanofiber within a nanofibrous mat aftercrosslinking.

FIG. 9B is a schematic illustration of the chemical structures ofcrosslinked alginate networks: network structures formed betweenalginate chains in nanofibers after crosslinking with (A) CaCl₂, (B)epichlorohydrin, (C) glutaraldehyde, (D) HMDI, and (E) ADA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides alginate-based nanofibers, scaffolds thatinclude nanofibers, implantable devices that include the nanofibers, andmethods for making the nanofibers by electrospinning.

In one aspect, the invention provides an alginate-based fiber. As usedherein, the term “alginate-based fiber” refers to a fiber that includesalginate. Alginate is a biodegradable polymer derived from seaweed.Alginate can be obtained from, for example, green algae (Chlorophyta),brown algae (Phaeophyta), and red algae (Rhodophyta). Alginate is alinear polysaccharide copolymer that consists of two stericallydifferent repeating units, (1→4)-α-L-guluronate (G unit) and(1→4)-β-D-mannuronate (M unit) in varying proportions. Alignate usefulin making the fibers of the invention has a mannuronic acid to guluronicacid (MG) ratio of 10 to about 90%. In some embodiments, alignate usefulin making the fibers of the invention has a MG ratio of about 50 to 70%.Alignate useful in making the fibers of the invention has a molecularweight of from about 10 kDa to about 1000 kDa. In some embodiments,alignate useful in making the fibers of the invention has a molecularweight of from about 50 kDa to about 500 kDa. Alignate useful in makingthe fibers of the invention has a viscosity of from about 50 to about600 cP (2% aqueous solution at 25° C.). In some embodiments, alignateuseful in making the fibers of the invention has a viscosity of fromabout 200 to about 400 cP (2% aqueous solution at 25° C.). Alginatebears structural resemblance to glycosaminoglycan (GAG), one of themajor components of the extracellular matrix (ECM) in human tissue.

In one embodiment, the fiber includes from about 30 to about 95 weightpercent alginate based on the total weight of the fiber. In oneembodiment, about 80 weight percent alginate based on the total weightof the fiber.

In one embodiment, the alginate fiber has a diameter of from about 20 toabout 2000 nm. In one embodiment, the fiber has a diameter from about 50to about 500 nm.

In addition to alginate, the alginate-based fiber of the inventionincludes a hydrophilic polymer to facilitate production of the fiber byelectrospinning. Hydrophilic polymers useful in making the fiber of theinvention include poly(alkylene oxide) polymers, polyvinyl alcoholpolymers, and polycarboxylic acid polymers. Representative poly(alkyleneoxide) polymers include block copolymers (for example, nonionic blockcopolymers commercially available under the designation PLURONIC and/orPOLOXAMER). In one embodiment, the poly(alkylene oxide) polymer ispolyethylene oxide. In one embodiment, the hydrophilic polymer (e.g.,poly(alkylene oxide)) has a molecular weight of from about 50 kDa toabout 1000 kDa. In one embodiment, the fiber includes from about 5 toabout 30 weight percent poly(alkylene oxide) based on the total weightof the fiber. In one embodiment, the fiber includes about 20 weightpercent poly(alkylene oxide) based on the total weight of the fiber.

To enhance structural integrity, the fibers and scaffolds of theinvention can be crosslinked. In one embodiment, the fiber of theinvention is crosslinked with an ionic crosslinking agent. Suitableionic crosslinking agents include a metal ions reactive toward alginatecarboxylic acid groups. Representative metal ions include calcium (2),barium (2), strontium (2), copper (2), zinc (2), magnesium (2),manganese (2), cobalt (2), lead (2), iron (3), and aluminum (3) ions.

The fiber of the invention can also be crosslinked with a covalentcrosslinking agent. Suitable covalent crosslinking agents includebifunctional crosslinking agents reactive toward alginate hydroxyland/or carboxylic acid groups. Representative covalent crosslinkingagents include carbodiimides, allyl halide oxides, dialdehydes,diamines, and diisocyanates. In certain embodiments, the covalentcrosslinking agent is selected from epichlorohydrin, gluteraldehyde,hexamethylene diisocyanate, adipic acid hydrazide, and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

In one embodiment, the fiber of the invention is both ionicallycrosslinked and covalently crosslinked.

In one embodiment, the fiber of the invention has a Young's modulus offrom about 0.004 to about 40 MPa.

In one embodiment, the fiber includes from about 30 to about 95 weightpercent alginate and from about 5 to about 70 weight percentpolyethylene oxide. In one embodiment, the fiber includes about 80weight percent alginate and about 20 weight percent polyethylene oxide.

In another aspect of the invention, fibrous scaffolds are provided. Thefibrous scaffolds of the invention include a plurality of the fibers ofthe invention, as described above. The scaffolds of the invention can bemanipulated to have shapes and sizes suitable for use as implantabledevices.

In one embodiment, the scaffold having a thickness of from about 0.05 toabout 5 mm.

The scaffolds can include one or more materials other than the fibers ofthe invention.

In one embodiment, the scaffold further includes chitosan. In thisembodiment, the scaffold includes from about 0.5 to about 15 weightpercent chitosan.

In one embodiment, the scaffold further includes collagen. In thisembodiment, the scaffold includes from about 0.5 to about 20 weightpercent chitosan.

In one embodiment, the scaffold further includes a biologically activeamino acid molecule. Representative amino acid molecules include growthfactors, fibronectin, laminin, vitronectin, integrins, nucleic acidmolecules, lipids, sugars, antisense molecules, anti-inflammatoryagents, immunomodulatory agents, molecules that promote cell migration,molecules that promote cell division, molecules that promote cellproliferation and differentiation, molecules that stimulate phenotypicmodification of cells, molecules that promote angiogenesis, moleculesthat promote vascularization, and molecules that promote extracellularmatrix disposition.

In one embodiment, the scaffold further includes a signaling ligand.Representative signaling ligands include members of the TGF-β family,bone morphogenic proteins, fibroblast growth factors-1 and -2,platelet-derived growth factor-AA and -BB, and platelet rich plasma andvascular endothelial cell-derived growth factor. In this embodiment, thescaffold includes from about 1 to about 100 ng signaling ligand per gramscaffold.

In a further aspect of the invention, implantable devices are provided.The implantable devices include the fibers and/or the scaffolds of theinvention described above. Representative implantable devices includecell transplantation devices, drug delivery devices, wound dressings andhemostats, surgically implantable devices for repairing damagedcartilage in mammalian articulating joints, fabric barriers tohyperplasia and tissue adhesion, vascular prostheses, nerve grafts,spinal fusion cages, and skin substitutes.

In another aspect, the invention provides a method for making thealginate fibers of the invention. In one embodiment, the method includes

(a) generating an electrostatic field between a first electrode and asecond electrode; and

(b) electrospinning an aqueous alginate solution comprising alginate anda hydrophilic polymer onto a collection surface located between thefirst electrode and the second electrode to provide a plurality ofalginate fibers on the collection surface.

In one embodiment of the method, the alginate solution includes fromabout 1 to about 8 percent by weight alginate. In one embodiment, thealginate solution includes from about 3 to about 5 percent by weightalginate.

In one embodiment, the hydrophilic polymer is selected from apoly(alkylene oxide), a polyvinyl alcohol, and a polycarboxylic acidpolymer. In one embodiment, the alginate solution includes from about 1to about 10 percent by weight poly(alkylene oxide). In one embodiment,the alginate solution includes from about 2 to about 4 percent by weightpoly(alkylene oxide). In one embodiment, the poly(alkylene oxide) ispolyethylene oxide.

In one embodiment, the ratio of alginate to poly(alkylene oxide) is fromabout 70:30 to about 95:5. In one embodiment, the ratio of alginate topoly(alkylene oxide) is about 80:20.

In one embodiment, the alginate solution further includes a co-solvent.Suitable co-solvents include polar aprotic solvents. Representativeco-solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile,dimethylformamide, and dimethylsulfoxide. In one embodiment, theco-solvent is dimethylsulfoxide. In one embodiment, the co-solvent ispresent in the solution in an amount from about 0.5 to about 20 weightpercent. In one embodiment, the co-solvent is present in the solution inan amount from about 1 to about 10 weight percent. In one embodiment,the co-solvent is present in the solution in about 5 weight percent.

In one embodiment, the alginate solution further includes a surfactant.Suitable surfactants include nonionic surfactants. Representativesurfactants include glycol stearate, cocoamide surfactants, andpolyoxyethylene isooctylphenyl ethers (Triton X-100 family). In oneembodiment, the surfactant is present in the solution in an amount fromabout 0.01 to about 1.0 weight percent. In one embodiment, thesurfactant is present in the solution in about 0.05 weight percent. Inone embodiment, the surfactant is polyoxyethylene(10) isooctylphenylether (Triton X-100).

In one embodiment, the alginate solution includes both a co-solvent anda surfactant, as described above.

In one embodiment, the zero shear viscosity of the alginate solution isfrom about 2.0 to about 8.0 Pa·s.

In one embodiment, the method further includes treating the plurality ofalginate fibers with an ionic crosslinking agent to provide a pluralityof ionically crosslinked alginate fibers. Suitable ionic crosslinkingagents are described above and include metal ions, such as calcium (2)ion, barium (2) ion, strontium (2), copper (2), zinc (2), magnesium (2),manganese (2), cobalt (2), lead (2), iron (3), and aluminum (3) ions.

In one embodiment, the method further includes treating the plurality ofionically crosslinked alginate fibers with a covalent crosslinking agentto provide a plurality of covalently crosslinked alginate fibers.Suitable covalent crosslinking agents are described above and includecarbodiimides, allyl halide oxides, dialdehydes, diamines, anddiisocyanates.

The fibers of the invention can be crosslinked ionically, covalently, orionically and covalently. In one embodiment, the method of the inventionincludes treating the alginate fibers with a covalent crosslinking agentto provide a plurality of covalently crosslinked alginate fibers.

Details of the alginate fibers of the invention, their properties, andmethods for their preparation are described below.

The present invention provides a method for fabrication ofalginate-based nanofibers using electrospinning. Althoughelectrospinning has proven to be an effective way of generatingnanofibrous structures for many materials, fabrication of alginatenanofibers by electrospinning is challenging. This is because thegelation of alginate solution starts to occur at very low polymerconcentrations (e.g., about 2 wt. % for alginate in deionized (DI)water). At such a low concentration, the solution contains insufficientmaterial to generate fibrous structures, and, rather, sprayed dropletsor a structure with short fibers embedded with beads is obtained. Atslightly higher polymer concentrations, the solution becomes so viscousthat it cannot be injected.

In the method of the invention, this problem is solved by theincorporation of a fraction of copolymer, and to apply one or moresurfactants or/and one or more cosolvents to the alginate solution. Theunderlying principle is to control the sol-gel transition by usingadditives, which interact with the alginate solution to reduce thesolution's viscosity, so that gelation can occur at a higher polymerconcentration.

In one embodiment, the copolymer useful in the method of the inventionis polyethylene oxide (PEO), a biocompatible and biodegradable polymer.PEO is a non-ionic polymer that is soluble in water, and interacts withalginate through hydrogen bonding, reducing the viscosity of thealginate solution. In one embodiment, alginate-PEO solutions wereprepared by mixing alginate and PEO in DI water, both at a fixedconcentration of 4 wt. %. Solutions with alginate/PEO ratios in therange 40:60-90:10 were prepared.

The instrumentation set-up for electrospinning alginate solutions wassimilar to that reported previously (N. Bhattarai, D. Edmondson, O.Veiseh, F. A. Matsen, M. Q. Zhang, Biomaterials 26:6176, 2005). Thedistance between two electrodes, that is, the distance between thesolution-injection tip and the fiber-collecting cylinder, was set to 20cm. A voltage of 15 kV was applied between the electrodes. Experimentalresults have shown that solutions with lower alginate/PEO ratios hadbetter electrospinnability, and stable, completely “bead-free”nanofibers were obtained at an alginate/PEO ratio of 50:50 or below. Asused herein, the term “electrospinnability” of a solution refers to theease with which electrospun fibers free from nonfibrous structures(sprayed droplets, fibers embedded with beads) are produced. To obtainalginate nanofibers at a higher alginate/PEO ratio, which is desirablefor some tissue-engineering applications, an amount of a surfactant(e.g., non-ionic surfactant such as polyoxyethylene(10) isooctylphenylether, commercially available under the designation Triton X-100) and/ora co-solvent (e.g., dimethyl sulfoxide (DMSO)) were introduced into thepolymer solution. Continuous nanofibers with alginate/PEO ratios in therange 70:30-90:10 were generated at typical concentrations of 0.5 wt. %Triton X-100 and 5 wt. % DMSO.

FIGS. 1A, 1A′, 1B, 1B′, 1C, and 1C′ are scanning electron microscopy(SEM) images of nanofibers spun from solutions with three differentalginate/PEO ratios, and the insets display the corresponding fiber sizedistributions. Solutions with alginate/PEO ratios in the range70:30-80:20 yielded cylindrical nanofibers with a mean diameter ofapproximately 75 nm and a relatively narrow size distribution. Thenanofibers spun from the solution with an alginate/PEO ratio of 90:10resulted in a relatively poor fiber size distribution.

Nanofibers spun from a solution with an alginate/PEO ratio of 80:20exhibited the most favorable morphology, while retaining a highalginate/PEO ratio, and, thus, these fibers were further studied fortheir structural integrity, mechanical properties, and cellularcompatibility. The choice of chondrocytes as target cells for thisinitial demonstration of cellular compatibility of alginate-basednanofibers was motivated by the inability of damaged articular cartilageto self-heal or be functionally restored, and the fact that the alginatematerial has been demonstrated to promote cartilage regeneration. TheECM of articular cartilage primarily consists of type II collagen andGAGs. Collagen and GAGs form a strong, porous, fibrous compositematerial. The collagen gives tensile strength to the cartilage, whereasthe GAG imparts elasticity. GAGs are considered to play a key role inmodulating chondrocyte cell morphology, differentiation, and function.

Stiffness, or modulus, is a physical material property to be consideredfor applications in regenerative medicine and tissue engineering. Inmost cases, it is preferable to have the material's modulus close tothat of the target tissue to avoid possible stress-shielding effects andmaintain sufficient mechanical support during in vitro and/or in vivocell growth and tissue-remodeling processes. Young's moduli ofrepresentative dried alginate-PEO nanofibers with and without calciumchloride crosslinkage are 5±1 MPa and 7±2 MPa, respectively, while a wetnanofiber crosslinked with calcium chloride has a modulus of 2.8±1.2 MPa(FIG. 2A). These values are comparable to those of human articularcartilage (1-10 MPa) and the equilibrium tensile modulus of bovinearticular cartilage (2-7 MPa).

For both in vitro and in vivo applications, materials are expected tomaintain their structural integrity in an aqueous environment. Thestructural integrity of alginate-based nanofibers in an aqueousenvironment has been examined by immersing alginate-PEO nanofibers indeionized (DI) water for up to 15 days. SEM images of the nanofibers atday one and day fifteen are shown in FIGS. 2B and 2C, respectively. Noapparent change in fiber morphology was observed, and the nanofibrousstructure was retained.

For the target tissue, cartilage chondrocyte-like cells (HTB-94) wereused to evaluate the cellular compatibility of alginate nanofibers.FIGS. 3A and 3B show the SEM images of chondrocyte cells cultured on thealginate-PEO nanofibers in Dulbecco's modified eagle medium (DMEM) for72 h. The cells attached well and formed cell clusters on thenanofibrous structure. The cells appeared round in shape and maintainedtheir characteristic phenotypes. A round cellular morphology isindicative of the characteristic function of chondrocytes, resultingfrom the organization of sparse active filaments. Cell viability wasassessed using a Live/Dead Assay Kit (Molecular Probes). Thefluorescence microscopy image of both live and dead cells on thenanofibrous scaffold incubated with chondrocyte cells for 72 hours isshown in FIG. 3C. The live cells appear as bright spots, and the deadcells are too few to be seen in the figure. The cellular viability,which is defined as the number of live cells divided by the total numberof cells (live cells plus dead cells), was determined to beapproximately 95%. Notably, unlike bulk alginate scaffolds, whichusually require precoated adhesion proteins such as fibronectin orarginine-glycine-aspatic acid peptides to facilitate cell adhesion, thealginate-based nanofibrous matrix does not need such pretreatment. Theunderlying mechanism for such an enhancement in cell adhesion is notclear, but the large specific surface areas of nanofibers that provide ahigh density of cellular binding sites, are thought to be one of themajor factors for the enhanced cellular activities.

Electrospinning of alginate solutions. Alginate solutions made bydissolving alginate in DI water at a concentration of 2 wt % or below donot generate a fibrous structure at any input voltage onelectrospinning. Instead, the solutions yielded droplets or leaked outfrom the injection tip. Increasing the alginate concentration from 2 to5 wt % caused the solution to become highly viscous, which impeded itscontinuous flow through the capillary tip, rendering the solutionunspinnable. Studies have shown that it is difficult, if not impossible,to produce nanofibers from aqueous solutions of pure natural polymers.In the method of the invention, alginate solutions are blended with amiscible hydrophilic synthetic polymers having neutral charge (e.g.,PEO). The hydrogen bonding between these hydrophilic polymers andnatural polysaccharides is responsible for the miscibility and improvedproperties of the blends solutions. To make alginate solution spinnable,PEO was used as the supporting polymer. Aqueous solutions of pure PEOare readily electrospinnable at a wide range of concentrations. Alginatesolutions (4 wt %) can be prepared at ambient conditions and so can PEOsolutions at the concentration.

In one embodiment, the alginate solution is a blend solution ofalginate/PEO at a final polymer concentration of 4 wt %. Blend solutionswith varying ratios of alginate to PEO from 40/60 to 90/10 were preparedby mixing a 4 wt % alginate solution with a 4 wt % PEO solution atdifferent proportions. As mentioned above, a 4 wt % pure alginatesolution is too viscous to be spinnable. Upon introducing PEO solutioninto the alginate solution, the viscosity of the resultant solution wasreduced, and electrospinning the solution yielded fibrous structures,suggesting that solution viscosity is one of determinants that regulatethe electrospinnability and the structures of produced nanofibers. Toquantify the effect of solution viscosity on the electrospun fiberstructure, the shear viscosity of the blend solutions as a function ofPEO/alginate ratio and shear rate was measured, and the electrospunproducts from these solutions were studied with SEM.

FIG. 4 is a graph comparing shear viscosities of polymer solutionshaving different alginate/PEO weight ratios (100:0; 90:10; 80:20; and60:40) as a function of shear rate. FIG. 4 shows that the viscosity ofthe blend polymer solution decreased monotonically with increasing PEOcontent. The SEM images of the as-spun products of alginate solutionsare presented in FIG. 5. FIGS. 5A-5D are SEM images of electrospunstructures from alginate solutions having alginate/PEO ratios of 90:10;80:20; 70:30; and 60:40, respectively. FIG. 5 shows that the as-spunproducts changed progressively from droplets, a structure of beads withshort fibers, to a fibrous structure as the PEO/alginate ratioincreased, corresponding to the decrease in solution viscosity shown inFIG. 4. Completely “bead-free” nanofibers were obtained at aPEO/alginate ratio equal to or greater than 50%.

The high viscosity of a pure alginate solution is believed primarily dueto strong intermolecular interaction between polymer chains and possibleentanglements that act as knots (i.e., the physical junctions ofalginate gels). The decrease in viscosity by addition of PEO can beattributed to the changes in inter- and intra-molecular interactions ofalginate chains. The introduced PEO acts as polybase and has strongtendency to form interpolymer complex with polycarboxylic acids. Theinterpolymer complexes have a compact structure, so that thehydrodynamic volume of the complex is smaller than the volume sum ofindividual macromolecules. Therefore, the complexation of two polymersin solution leads to a decrease in viscosity. The addition of PEOmolecules into the polyanionic alginate solution creates interpolymercomplexes by forming hydrogen bonds between hydrogen onhydroxyl/carboxyl groups of alginate and ether oxygen on PEO, whichdisrupts the associative structure of alginate chains.

Both PEO and alginate are biocompatible and biodegradable polymers andhave been used in a variety of tissue engineering applications. However,natural polymers are generally preferable in regenerative medicine. Itis therefore advantageous to produce an alginate fiber product that hasthe alginate component as great as possible in produced nanofibers whilemaintaining a uniform fibrous. As illustrated in FIGS. 4 and 5, moreuniform nanofibrous structures are achieved at an alginate/PEO ratioless than about 60/40.

To reduce the alginate solution viscosity while maintaining a highalginate/PEO ratio, in one embodiment, the alginate solution includes anonionic surfactant.

FIGS. 6A-6D are images of the electrospun structures when 0.5 wt %nonionic surfactant (Triton X-100) was introduced in 4 wt % solution ofalginate/PEO (80/20). The addition of the surfactant substantiallyimproved the fibrous structure compared to the solution with the samealginate/PEO ratio without the surfactant (FIG. 5B). However, theas-spun structure was not exclusively fibrous and “beads” were seenembedded in the fibers. To further improve the electrospun structure, aco-solvent (DMSO) was introduced into the solution. A bead-free, uniformfibrous structure was produced by addition of surfactant (0.5 wt %Triton X-100) and co-solvent (5 wt % DMSO). The resultant structures areshown in FIGS. 6B-6D (differing magnifications). By the method, solid,cylindrical nanofibers with a main diameter of about 75 nm were obtainedand no phase separation between alginate and PEO was observed.

FIGS. 6A-6D are images of electrospun alginate/PEO fibers from 80:20alginate/PEO solutions: FIG. 6A is a SEM image of fibers obtained byadding Triton X-100 (0.5 wt %); FIG. 6B is a SEM image of fibersobtained by adding Triton X-100 (0.5 wt %) and DMSO (5.0 wt %); FIG. 6Cis a transmission electron microscope (TEM) image of the fibers of FIG.6B; and FIG. 6D is a higher magnification TEM image of the fibers ofFIG. 6B.

Triton X-100, a nonionic surfactant, is structurally similar to PEO,bearing short chain of ethylene oxide repeating units in its hydrophiliclong tail. The surfactant dissolves readily in aqueous solution andinteracts with alginate in a fashion similar to those between alginateand PEO. When DMSO was used as the co-solvent, a dipole-dipoleinteraction was introduced between alginate chains and DMSO because DMSOhas a large dipole moment (i.e., 3.96). This interaction weakened theassociation between alginate chains and improved the solutionspinnability. As demonstrated, the combination of a co-solvent (e.g.,DMSO) and a nonionic surfactant (e.g., Triton X-100) affordedsignificant processing advantages to modulate the alginate solution athigh alginate/PEO ratios. The incorporation of small amounts of TritonX-100 and DMSO into alginate/PEO solutions has no effect on thechemistry of the final fibrous product because both are readily solublein water and can be easily removed from nanofibers during the washing orcrosslinking of the nanofibers. Additionally, as a co-solvent, most ofthe DMSO evaporates during the electrospinning as the solution jettravels from the injection tip to the collector.

Effect of solution viscosity and stability on the formation ofnanofibers. The properties of alginate solutions appear to change duringstorage over time in the ambient environment. Solution viscosity playsan important role in electrospinnability of polymer solutions and thestructures of resultant polymer fibers.

Freshly prepared alginate/PEO solution were observed to be difficult toprocess. However, when the solution was left in the ambient environmentfor certain time, the solution became more spinnable. Therefore, it isbelieved that the underlying mechanism for this observation providesinsight into electrospinnability of the polymer solutions. Spinnabilityappears to be directly related to solution viscosity. To determinewhether the viscosity of an alginate polymer solution would change overtime at ambient conditions, a 4 wt % alginate solution was maintained atroom temperature over a period of 30 days and measuring the viscosity ofthe solution at different time points. As shown in FIG. 7A, theviscosity of the solution dropped considerably in the first two weeksand gradually saturated thereafter. The viscosity drop with the storagetime was believed attributable to a decrease in the average molecularweight of alginate as a result of polymer chain degradation. Alginatemolecular chain scission was caused by the breakage of glycosidic bondsdue to aqueous hydrolysis. Chain scission was confirmed by maintainingan alginate solution in the ambient environment and then sampling thesolution at different storage times and characterizing the samples byFTIR and UV spectroscopy. FIG. 7B shows the FTIR spectra of thesesamples, with the spectrum of as-received alginate powder (as control)shown for comparison. Peaks of sodium alginate at 3400, 1610 and 1089cm⁻¹ are attributed to —OH, C—O—O⁻, C—O—C groups, respectively. Thebreakage of glycosidic bonds leads to the formation of more hydroxylgroups, which is characterized by a sharp increase in the intensity ofthe hydroxyl group peak at 3400 cm⁻¹ over storage time as compared tothe broad and weak absorbance of the as-received alginate. The increasedpeak intensities of C—O—C and C—O—O⁻ groups are also as expected asshown in FIG. 7B. The alginate polymer chain degradation was furtherconfirmed by UV spectrometry and the results are shown in FIG. 7C. TheUV absorption band was shifted from 225 nm at day 0 to 240 nm at day 15and the intensity of the absorbance increased with solution storagetime. The peak shift and increase in absorbance are due to the doublebonds formed upon a polymer main chain scission creating a ring opening,as also observed in radiation induced degradation of alginate. Thetime-dependent changes in both FTIR and UV spectra correspond well tothe change in solution viscosity: the most significant changes in thespectra occurred in the first two weeks as the solution viscosity (seeFIG. 7A). FIG. 7A is a graph illustrating zero shear viscosity of a 4 wt% alginate solution as a function of storage time. FIG. 7B compares theinfrared (FTIR) spectra of the 4 wt % alginate solution as a function ofstorage time (Day 6, 10, 15, 20, and 30) to the infrared spectrum ofas-received alginate powder (Control). FIG. 7C compares the ultraviolet(UV) absorbance spectra of the 4 wt % alginate solution as a function ofstorage time (Day 6, 10, 15, 20, and 30) to the absorbance spectrum ofas-received alginate powder (Control).

The influence of solution storage time on electrospun product wasevaluated. Alginate solutions were maintained in the ambient environmentfor different times were mixed with PEO solutions to prepare blendsolutions with an alginate/PEO ratio of 80/20. These blend solutionswere then electrospun immediately after preparation and the resultantstructures were examined by SEM. The study revealed that alginatesolutions subjected to different storage times can be divided into threeregions in term of viscosity as shown in FIG. 7A, with the solutionsthat fall in the same region producing similar fiber morphology. FIGS.7D-7F show representative images of the electrospun fibrous structurefor each region (alginate solutions stored for 2, 5 and 15 days). FIGS.7D-7F are SEM images of fibers electrospun from alginate solutionshaving viscosities in Regions I, II, and III, respectively, of FIG. 7A.

These results further demonstrated a strong correlation between solutionviscosity and electrospun structure. Solutions having viscosities thatfall in Region II produced desired uniform nanofibrous structures.Solutions stored for prolonged time beyond 15 days had too low viscosity(Region III) and yielded “bead-string” structures. The solution storagetime also affected the electrospinning rate. The electrospinning ratewas measured by solution volume consumption in unit time. For solutionsstored for 2, 5 and 15 days the rates were found to be 0.5±0.15, 5.0±0.8and 15.0±2.5 ml/h, respectively.

A practical implication of this finding is that as-received alginate isnot necessarily suitable for fabrication of nanofibers byelectrospinning even if the polymer has been demonstrated to bespinnable. Thus, the solution viscosity rather than storage time may betaken as a practical indicator to identify the spinnability of aparticular polymer. Changing molecular weight through storage or a“natural” course is not a recommended approach to reduce solutionviscosity.

Integrity of nanofibrous structure in aqueous media. Alginate and PEOare soluble in aqueous medium. Most tissue engineering applicationsrequire materials to maintain structural integrity in aqueousenvironments (e.g., cell culture medium, human body) for prolongedperiods. This is particularly important for nanofeatured materials asthey degrade more quickly than bulk materials. One way to improve thestability of hydrophilic polymeric materials in solution is to crosslinkthe materials. Crosslinking may be applied to each fiber component(alginate and PEO). In one embodiment, the fibers are crosslinked byalginate crosslinking alginate. Alginate crosslinked with ioniccrosslinking agents are stable in water, but unstable in solutionscontaining salt ions. For both in vitro and in vivo applications,materials are often exposed to aqueous media with different ionicstrengths. In this case, covalent crosslinking of nanofibers may provebe a better solution. However, covalent crosslinking of alginatenanofibers needs to proceed in aqueous solution, a process that itselfmay degrade alginate nanofibers within the reaction time frame. In oneembodiment of the method of the invention, the fibers are firstionically crosslinked and then covalently crosslinked.

For ionic crosslinking, nanofibrous mats were soaked with ethanol andthen with saturated solution of calcium chloride (CaCl₂) in 75% ethanol,followed by incubation in aqueous CaCl₂ solution. The structuralstability of the ionically crosslinked nanofibrous mats was examined byincubation in DI water and SBF. FIGS. 8A and 8B are SEM images of arepresentative ionically (calcium chloride) crosslinked alginate(alginate/PEO 80:20) fibrous mat of the invention incubated in DI water(FIG. 8A) and SBF (FIG. 8B) for seven days at 37° C. FIG. 8A shows thatthe ionically crosslinked alginate fibrous mat remained intact in water,whereas FIG. 8B shows that the mat completely lost its initialnanofibrous structure. A similar result as in SBF was obtained for themat incubated in PBS.

The crosslinking of alginate based nanofibers with bivalent metal ionssuch as Ca²⁺, Ba²⁺, Sr²⁺ is via electrostatic interaction of twocarboxyl groups on different alginate chains, in which the polymericmolecule loses its freedom of movement and a crosslinked networkstructure is formed. The crosslinked alginate structure is stable inwater in a temperature range of 0-100° C. However, when placed in SBF,which contains salt ions Na⁺, the dissolution of the nanofiber matoccurs as a result of the exchange of Na⁺ ions in the solution with Ca²⁺ions bound to carboxylates in alginate chains. For cell culture medium,this may be less problematic, because the ionic salt concentration inin-vitro cell culture medium is maintained at a level lower than in SBFand PBS where the ion exchange rate is low and the nanofibrous structurecan survive prolonged culture time.

In order to retain structural integrity of alginate based nanofibrousmats in aqueous media of a wide range of ionic strengths, thenanofibrous mats crosslinked with CaCl₂ were further covalentlycrosslinked. Covalent crosslinking of alginate is possible becausealginate has two active functional groups (i.e., hydroxyl andcarboxylate). Four representative homobifunctional crosslinking agentswere used to crosslink the alginate fibers: (1) epichlorohydrin, (2)glutaraldehyde, (3) hexamethylene diisocyanate (HMDI), and (4) adipicacid hydrazide (ADA). Each of these crosslinking agents is used tocrosslink polymers bearing hydroxyl and carboxylic functional groups torender water-soluble polymers water insoluble.

FIGS. 8C-8F are SEM images of representative crosslinked alginate(alginate/PEO 80:20) fibrous mats of the invention incubated in SBF (pH7.4) for seven days at 37° C.: the fibrous mats were first ionicallycrosslinked with calcium chloride followed by covalent crosslinking withepichlorohydrin (FIG. 8C), glutaraldehyde (FIG. 8D), hexamethylenediisocyanate (HMDI) (FIG. 8E), or adipic acid hydrazide (ADA) (FIG. 8F).

The process of ionic and covalent crosslinking, as well as the resultantassociations between polymer chains in a single nanofiber within ananofibrous mat after crosslinking is illustrated schematically in FIG.9A. The chemical structures of the resultant crosslinked networks areillustrated schematically in FIG. 9B; molecular network structuresformed between alginate chains in nanofibers after crosslinking with (A)CaCl₂, (B) epichlorohydrin, (C) glutaraldehyde, (D) HMDI, and (E) ADA.The covalent crosslinking of alginate nanofibers occurs by the formationof inter- and intra-molecular covalent bonds between alginate chains inthe nanofiber as well as at fiber/fiber overlap junctions.Epichlorohydrin, glutaraldehyde, and HMDI utilize the hydroxyl groups ofalginate, whereas ADA utilizes carboxyl groups. Covalently crosslinkednanofibrous mats were characterized by dissolution test in PBS. FIGS.8C-8F show SEM images of nanofibrous mats crosslinked withepichlorohydrin, glutaraldehyde, HMDI, and ADA, respectively, afterincubated in SBF for one week.

In contrast to ionically crosslinked nanofibrous mats (e.g., FIG. 5B),the mats after dual crosslinking (i.e., both ionic and covalent)retained their fibrous structures, although changes of different degreesin fiber morphology (swelling or melting) were observed except for thefibers crosslinked with ADA which showed no evident change.

Because the solubility of epichlorohydrin in water is relatively poor,the epichlorohydrin crosslinking reaction was conducted in 50% ethanol.To promote the reaction of epichlorohydrin with hydroxyl groups ofalginate, the pH of the reaction mixture was maintained basic byaddition of dilute NaOH solution. For crosslinking alginate nanofibersusing glutaraldehyde, the reaction was carried out in 50% ethanol andcatalyzed with dilute HCl to facilitate the reaction between aldehydeand the hydroxyl groups. The observed fiber swelling and melted fiberjunctions in PBS after crosslinking is common for hydrophilicbiopolymers when they are incubated in aqueous medium. Additionally,both epichlorohydrin and glutaraldehyde crosslinking reactions occurredat highly basic or acidic solutions, which may potentially damage thefine structures of the nanofibrous mats during the crosslinking.Crosslinking alginate nanofibers with HMDI in DMSO/DMF mixture avoidsthe use of aqueous solution. However, the resultant nanofibrous matexhibited relatively poor morphology in PBS and some of the nanofibersappeared dissolved after one week of incubation (FIG. 8E). This islikely due to less efficient crosslinking as a result of the poorsolubility of alginate in DMSO/DMF mixture. The ADA crosslinking wascarried out in MES aqueous solution at mild pH and the reaction time wasrelatively short. This approach yielded the best retained fibrousstructure in PBS (FIG. 8F) and used no organic solvents forcrosslinking, which is more favorable for a majority of tissueengineering applications.

The crosslinking of representative alginate fibrous mats of theinvention is described in Example 2.

The alginate fibers of the invention are fabricated by electrospinning.Polymer solution viscosity was a key factor that regulates thespinnability and the structure of the electrospun product. Solutionshaving certain ranges of viscosity produce fibrous structures. Solutionviscosity can be changed in a number of ways including addition of asecond hydrophilic polymer and/or surfactants or alteration of polymermolecule weight. Alginate molecular structure in solution and thus thesolution viscosity changes over time even in the ambient environmentresulting in inconsistent structural properties in polymeric nanofibrousmaterial production, as nanofeatured materials are particularlysusceptible to changes in molecular structure, environmental conditions(e.g., heat or radiation), and experimental parameters. Polymer solutionviscosity serves a practical indicator to identify the spinnability of apolymer solution and differentiate the structures of electrospunproducts.

For tissue engineering applications, a scaffolding material must retainits structural integrity during time of service (from days to monthsdepending on application) in designated biological environments whichcan be in vitro or in vivo. The crosslinked alginate fibers of theinvention improve the stability of alginate based nanofibers indifferent chemical or biological environments.

The following examples are provided for the purpose of illustrating, notlimiting, the invention.

EXAMPLES Example 1 The Preparation and Characterization ofRepresentative Alginate Fibers

In this example, the preparation and characterization of representativealginate fibers of the invention, alginate-PEO fibers, is described.

Materials. Alginic acid sodium salt from brown algae with mediumviscosity and PEO (weight-average molecular weight (Mw) 900 kDa) werepurchased from Sigma-Aldrich. Triton X-100 was purchased from VWR. Ahuman chondrocyte-like cell line (HTB-94) was obtained from AmericanType Culture Collection (Manassas, Va.). All culture media and reagentswere purchased from Invitrogen Life Technologies. Two different buffers,phosphate-buffered saline (PBS) and simulated body fluid (SBF), wereprepared in the lab using buffer constituents from Sigma and J. T. BakerChemical Co.

Electrospinning of Nanofibers. 4 wt % alginate solutions and 4 wt % PEOsolutions were prepared separately by dissolving alginate or PEO in DIwater, followed by centrifugation to remove air bubbles. The alginateand PEO solutions of different proportions were then mixed to obtainmixtures with weight ratios of alginate to PEO in the range 40:60-90:10,and the resultant mixtures were stirred for 3 h. Solutions containing0-1.0 wt. % Triton X-100 and 0-10 wt. % DMSO were mixed withalginate-PEO solutions, and the mixtures were stirred for an additional3 h and centrifuged to remove air bubbles before use in electrospinning.The electrospinning system was similar to that reported previously (N.Bhattarai, D. Edmondson, O. Veiseh, F. A. Matsen, M. Q. Zhang,Biomaterials 26:6176, 2005). Briefly, a DC voltage of 10-15 kV with lowcurrent output (High DC power supply, Del Electronics Corp.) was appliedbetween the syringe tip and a cylindrical collector. The typicaldistance between the syringe tip and the grounded collector was 17-20cm. Polymer solution inside the syringe was charged with a positivevoltage by dipping a platinum wire into the solution from a positivelead; the cylindrical collector was grounded.

Characterization of the Nanofibrous Structure. The electrospunnanofibers were collected as a fibrous mat from the collector. Thenanofibrous structure was crosslinked with CaCl₂ solution at roomtemperature. The nanofibrous mat was soaked in 95% ethanol for 5 min andrinsed with 1 wt. % CaCl₂ solution in ethanol for 10 min. Theethanol/CaCl₂-treated fiber mat was incubated with a CaCl₂ solution for1 h. The nanofibrous mat was then immersed in DI water for 1 h andrinsed with DI water to remove excess CaCl₂. Samples were dried at roomtemperature after washing with absolute ethanol. To examine theintegrity of the nanofibers, the nanofibrous mat was immersed in DIwater at room temperature for specified time periods. The morphology ofthe nanofibrous structure was examined using scanning electronmicroscopy (SEM) (JEOL JSM-7000) at an accelerating voltage of 10 kVafter sputter-coating the specimens with Au—Pd. The average diameter ofthe electrospun nanofibers was determined by measuring the diameters ofthe nanofibers at 100 different points in a 645×484 SEM image.

FIGS. 1A-1C′ are scanning electron microscope (SEM) images ofrepresentative alginate-polyethylene oxide (PEO) fibers of the inventionelectrospun from alginate/PEO solutions containing 0.5 wt % Triton X-100surfactant and 5 wt % DMSO co-solvent: FIG. 1A is an image of arepresentative alginate-PEO fiber spun from a solution having analginate/PEO ratio of 70:30; FIG. 1A′ is a higher magnification image ofthe image of FIG. 1A; FIG. 1B is an image of a representativealginate-PEO fiber spun from a solution having an alginate/PEO ratio of80:20; FIG. 1B′ is a higher magnification image of the image of FIG. 1B;FIG. 1C is an image of a representative alginate-PEO fiber spun from asolution having an alginate/PEO ratio of 90:10; and FIG. 3A′ is a highermagnification image of the image of FIG. 3A;

Both dried and wet nanofibrous mats were evaluated for a stress-strainresponse using a microtensile testing machine designed for high-accuracysmall-sample testing. Wet samples were prepared by incubating CaCl₂crosslinked nanofibrous mats in DI water for 1 h at 37° C. immediatelyprior to the tensile test. The load cell had a loading range of ±30 gwith an incremental accuracy of 0.001 g. The data were acquired througha computer interface connected to a load transducer (Model SS-2) and anelectronic signal conditioner (PTC Electronics, Wyckoff, N.J.) on thetest machine. The load was applied by a stepper motor system (MotionGroup, Clovis, Calif.) controlled by a LabView routine written by theauthors. Nanofibrous mats were cut in a rectangular shape with across-section of 60 mm². The tensile modulus was calculated from thestress-versus-strain curve.

FIG. 2A is a bar graph comparing the tensile moduli of representativealginate-PEO fibers of the invention electrospun from an 80:20alginate/PEO solution: as spun fiber; calcium chloride crosslinked driedfiber; and calcium chloride crosslinked wet fiber.

Cell Culture. The nanofibrous mats crosslinked with calcium chloridewere mounted on cover glass (22 mm²) and sterilized with ethylene oxidegas. The nanofibrous mats were fixed on glass coverslides using coppertapes (22 mm²) and washed several times with 75% ethanol and DI water atneutral pH to remove residual solvents. 10⁵ chondrocyte (HTB-94) cellsin 1 mL DMEM containing 10% fetal bovine serum, 50 IU mL⁻¹ penicillin,and 50 μg mL⁻¹ streptomycin were then seeded on each nanofibrous mat in24-well culture plates. Cellular constructs were harvested after 3 days,washed extensively with PBS, and fixed with Karnovsky's fixativeovernight at room temperature. All the samples were gradually dehydratedwith 50, 75, 95, and 100% ethanol, for 2 h each. The samples were thendried using a critical-point dryer and coated with Au—Pd for SEMimaging.

FIGS. 2B and 2C are scanning electron microscope (SEM) images ofrepresentative alginate-polyethylene oxide (PEO) fibers of the inventionelectrospun from an 80:20 alginate/PEO solution after immersion indeionized (DI) water for 1 day and 15 days, respectively.

Cell Viability. Cell viability was assessed using a Live/Dead Assay Kit(PA-3016, Molecular Probes). 100-150 μL of the Live/Dead assay reagentwas added to cell-cultured alginate nanofibers on a glass coverslip (22mm²), and the coverslip was incubated for 45 min. The reagent containedthe vital dye calcein-AM and nuclear stain ethidium homodimer-1 (bothfrom the Live/Dead Assay Kit). Calcein-AM is a non-fluorescent,cell-permeant fluorescein derivative, which is converted by cellularenzymes into cell-impermeant and highly fluorescent calcein. Calceinaccumulates inside live cells with intact membranes and renders thecells fluorescent green. Ethidium-homodimer-1 enters dead cells withdamaged membranes and undergoes a 40-fold enhancement of fluorescenceupon binding to their DNA, causing the nuclei of dead cells to fluorescered. This double-staining showed both live and dead cells attached onthe scaffold. The wet coverslip was inverted and placed on themicroscope slide. Images of chondrocyte cells on nanofibers wereacquired using an inverted fluorescent microscope equipped with acharge-coupled device (CCD) camera. Cellular viability was quantifiedfollowing the manufacturer's instruction.

FIGS. 3A-3C compare the in vitro cellular compatibility of alginatefibers electrospun from an 80:20 alginate/PEO solution: FIG. 3A is ascanning electron microscope image of chondrocytes grown on alginate-PEOfibers; FIG. 3B is a higher magnification image of the image of FIG. 3B;and FIG. 3C is a fluorescence image of cells on fibers with theLive/Dead cell stain.

Example 2 The Preparation and Characterization of RepresentativeCrosslinked Alginate Fibers

In this example, the preparation and characterization of representativealginate fibers of the invention, crosslinked alginate-PEO fibers, isdescribed.

Materials. Alginic acid sodium salt from brown algae having mediumviscosity was purchased from Sigma-Aldrich chemical Co. Based on thesupplier's information, the alginate with viscosity about 350 cP (2%solution, 25° C.) is polyuronic acid composed primarily of linear andhydrophilic anhydro-β-D-mannuronic acid residues with 1→4 linkage.Polyethylene oxide (PEO) (Mw=900 kDa), epichlorohydrin, glutaraldehyde,hexamethylene diisocyanate (HMDI), 2-(N-morpholino)ethanesulfonic acidhydrate (MES) buffer, 1-hydroxybenzotriazole (HOBt),1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and adipic acidhydrazide (AAD) were also obtained from Sigma-Aldrich Chemical Co.Triton X-100 was purchased from VWR Co. Two different buffers, phosphatebuffered saline (PBS) and simulated body fluid (SBF), were prepared inthe lab using buffer constituents purchased from Sigma and J. T. BakerChemical Co. SBF with an ion concentration proximately equal to that ofhuman blood plasma was prepared by dissolving reagent grade NaCl,NaHCO₃, KCl, K₂HPO₄.3H₂O, MgCl₂.6H₂O, CaCl₂, and Na₂SO₄ in distilledwater. They were buffered at pH 7.4 with tris(hydroxymethyl)aminomethane((CH₂OH)₃CNH₂) and hydrochloric acid.

Preparation of the Electrospinning Solution. 4 wt % alginate solutionsand 4 wt % PEO solutions were first prepared separately by dissolvingalginate and PEO in deionized water, followed by centrifugation toremove air bubbles. Alginate and PEO solutions of different proportionswere then mixed to obtain the blend solutions with weight ratios ofalginate to PEO ranging from 40:60 to 90:10, and the resultant solutionswere stirred for 3 h. Solutions containing 0-1.0 wt % of Triton X-100and 0-10 wt % of dimethylsulfoxide (DMSO) were mixed with alginate/PEOsolutions, and the mixtures were stirred for additional 3 h andcentrifuged to remove air bubbles before use.

Electrospinning of Fibers. The electrospinning process used in thisstudy was as described above in Example 1. Briefly, the solution was fedinto a 3 ml disposable syringe fitted with a pipette tip of 0.5 mm indiameter. A DC voltage of 10-15 kV (High DC power supply, DelElectronics Corp.) was applied between the syringe tip and a cylindricalcollector covered with aluminum foil. The cylinder had a diameter of 7cm and was driven by a DC motor with controllable speed. The typicaldistance between the syringe tip and the grounded collector was 17-20cm. During the spinning process, the pendant droplet at the syringe tipwas split by a repulsion force set by the charge in the droplet, andformed a jet of a cone-like shape traveling towards the collector,during which time the solvent evaporated and polymer fibers deposited onthe collector in form of a nonwoven fibrous mat. All the spinningexperiments and drying of as-spun nanofibers were conducted at roomtemperature. The diameter, morphology, and surface topography of thenanofibers were examined using SEM (JEOL JSM-840A) at an acceleratingvoltage of 10 kV. Electrospun nanofibers were sputter-coated with Au/Pdprior to SEM analysis. The mean diameter of electrospun nanofibers wasdetermined by measuring the diameters of nanofibers at 100 differentpoints in a 645×484 SEM image. A Philips CM 100 transmission electronmicroscope (TEM) was used to observe internal features of nanofibers.For TEM observation, a thin fibrous membrane was sandwiched in PELCOfolding grids and images were acquired at the edges of the membrane. Asthe intrinsic difference in electron density between alginate and PEOpolymers provided adequate contrast, no staining was needed.

FIGS. 6A-6D are images of electrospun alginate/PEO fibers from 80:20alginate/PEO solutions: FIG. 6A is a SEM image of fibers obtained byadding Triton X-100 (0.5 wt %); FIG. 6B is a SEM image of fibersobtained by adding Triton X-100 (0.5 wt %) and DMSO (5.0 wt %); FIG. 6Cis a transmission electron microscope (TEM) image of the fibers of FIG.6B; and FIG. 6D is a higher magnification TEM image of the fibers ofFIG. 6B.

Characterization of Stability of Alginate Solution. The shearviscosities of the solutions of different alginate/PEO ratios weremeasured in a shear rate range of 5-500 s⁻¹ using a Haake Viscometer(VT550) equipped with double concentric cylinder-type SP2P sensors. FTIRspectroscopy and UV-VIS spectroscopy were utilized to identify thedecrease in molecular weight and change in molecular structure ofalginate in aqueous solution over storage time, which directly affectsthe spinnability of the solution. For measurements of changes insolution viscosity over a time course, the solution was prepared asdescribed above and maintained at room temperature. A 10 ml sample wasperiodically taken out from the solution and the samples collected atdifferent times were stored in a freezer at −20° C. Viscosity of eachsample was measured immediately after defrosting the sample. For UV andFTIR experiments, powdered alginate samples were first prepared byprecipitating the alginate solutions collected at different times withethanol. The samples were washed with a mixture of ethanol/water (80/20v/v) and subsequently dried under vacuum. For UV measurements, 0.5 wt %alginate solutions in DI water were prepared and their UV absorptionspectra were acquired using a UV-Vis Spectrophotometer (Hewlett Packard8452A Diode Array) operating at 200-400 nm with 2 nm resolutions. ForFTIR analysis, samples were prepared by mixing alginate powder with KBrat alginate/KBr w/w ratio of 1/60 using a macro KBr die kit. The solidpellet was placed in a magnetic holder, and the system was purged withnitrogen before testing. Polarized FTIR spectra of 200 scans at 4 cm⁻¹resolutions were obtained using a Nicolet 5DX spectrometer equipped witha DTGS detector and a solid transmission sample compartment. Spectrumanalysis and display were performed using standard Nicolet and MicrocalOrigin software.

FIG. 7A is a graph illustrating zero shear viscosity of a 4 wt %alginate solution as a function of storage time. FIG. 7B compares theinfrared (FTIR) spectra of the 4 wt % alginate solution as a function ofstorage time (Day 6, 10, 15, 20, and 30) to the infrared spectrum ofas-received alginate powder (Control). FIG. 7C compares the ultraviolet(UV) absorbance spectra of the 4 wt % alginate solution as a function ofstorage time (Day 6, 10, 15, 20, and 30) to the absorbance spectrum ofas-received alginate powder (Control).

Crosslinked Fibrous Mats. Electrospun nanofibrous mats were firstcrosslinked using CaCl₂ solution at room temperature and then optionallycrosslinked using one of four methods described below. Nanofibrous matswere immersed in ethanol for 5 min and then in saturated solution ofCaCl₂ in 75% ethanol for 10 min. The samples were then incubated in anaqueous CaCl₂ solution for 1 h and immersed in DI water for additional 1h, followed by rinsing with excess DI water to remove unreacted CaCl₂.After washing with absolute ethanol, the samples were dried at roomtemperature.

Epichlorohydrin Crosslinking. Dried nanofibrous mats were soaked in asuspension containing 5M epichlorohydrin and 0.1M NaOH in 50% ethanol.The crosslinking reaction was allowed to proceed for 24 h and stopped byaddition of 5M HCl. Crosslinked nanofibrous mats were washed with 75%ethanol and then with excess DI water. The samples were dried underreduced pressure at room temperature.

Glutaraldehyde Crosslinking. Nanofibrous mats were immersed in 50 wt %ethanol containing 250 mM of glutaraldehyde and 0.05M HCl. Thecrosslinking reaction was allowed to proceed at room temperature for 48h. Upon the reaction completion, nanofibrous mats were washed withexcess DI water and dried under reduced pressure at room temperature.

Hexamethylene Diisocyanate (HMDI) Crosslinking. Dried nanofibrous matswere soaked in and rinsed with a DMSO/DMF (1:1 v/v) mixture. A mixtureof 5 wt % solution of hexamethylene diisocyanate (HMDI) and 0.5 g oftriethylamine (TAE) was prepared in DMSO/DMF (1:1 v/v) and poured on thetop of the fiber mat. The crosslinking reaction was allowed to proceedat room temperature for 48 h. Excess reactants and byproducts werewashed away from samples by extracting with tetrahydrofuran (THF) for 5h using a Sohxlet apparatus, and the samples were dried at 40° C. undervacuum for 24 h.

Adipic Acid Hydrazide (ADA) Crosslinking. Nanofibrous mats were soakedin 2-morpholinoethanesulfonic acid (MES) buffer (at pH 6.5) containing 5mM 1-hydroxybenzotriazole for 2 h, and 5 mM EDC was then added into themixture. After the samples were soaked in the mixture for additional 1h, 1M AAD was added. The crosslinking reaction was allowed to proceedfor 20 h at room temperature on a rocking shaker, and upon completion ofthe reaction the samples were washed with excess DI water. Thenanofibrous mats were then dried under reduced pressure.

To test the stability of nanofibrous mats in aqueous ionic environmentsover time, crosslinked nanofibrous mats were incubated in SBF (pH=7.5)at room temperature. Samples were collected periodically from theincubated solution and dried at room temperature after three washes withDI water/ethanol mixture. Similar experiment was also performed byincubating the crosslinked nanofibrous mat in PBS (pH=7.5). Theintegrity of alginate nanofibers were examined by morphological changesobserved under SEM.

FIGS. 8A and 8B are SEM images of a representative ionically (calciumchloride) crosslinked alginate (alginate/PEO 80:20) fibrous mat of theinvention incubated in DI water (FIG. 8A) and SBF (FIG. 8B) for sevendays at 37° C.

FIGS. 8C-8F are SEM images of representative crosslinked alginate(alginate/PEO 80:20) fibrous mats of the invention incubated in SBF (pH7.4) for seven days at 37° C.: the fibrous mats were first ionicallycrosslinked with calcium chloride followed by covalent crosslinking withepichlorohydrin (FIG. 8C), glutaraldehyde (FIG. 8D), hexamethylenediisocyanate (HMDI) (FIG. 8E), or adipic acid hydrazide (ADA) (FIG. 8F).

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A fiber, comprising alginate and a hydrophilic polymer.
 2. The fiberof claim 1 having a diameter of from about 20 to about 2000 nm.
 3. Thefiber of claim 1, comprising from about 30 to about 95 weight percentalginate based on the total weight of the fiber.
 4. (canceled)
 5. Thefiber of claim 1, wherein the hydrophilic polymer selected from thegroup consisting of a poly(alkylene oxide), a polyvinyl alcohol, and apolycarboxylic acid polymer.
 6. The fiber of claim 5, wherein thepoly(alkylene oxide) is polyethylene oxide.
 7. The fiber of claim 5,wherein the poly(alkylene oxide) has a molecular weight of from about 50kDa to about 1000 kDa.
 8. The fiber of claim 5, comprising from about 5to about 30 weight percent poly(alkylene oxide) based on the totalweight of the fiber.
 9. (canceled)
 10. The fiber of claim 1 crosslinkedwith an ionic crosslinking agent.
 11. The fiber of claim 10, wherein theionic crosslinking agent is a metal ion selected from the groupconsisting of calcium (2), barium (2), strontium (2), copper (2), zinc(2), magnesium (2), manganese (2), cobalt (2), lead (2), iron (3), andaluminum (3) ions.
 12. The fiber of claim 10 further crosslinked with acovalent crosslinking agent.
 13. The fiber of claim 12, wherein thecovalent crosslinking agent is a bifunctional crosslinking agentselected from the group consisting of carbodiimides, allyl halideoxides, dialdehydes, diamines, and diisocyanates.
 14. The fiber of claim12, wherein the covalent crosslinking agent is selected from the groupconsisting of epichlorohydrin, gluteraldehyde, hexamethylenediisocyanate, adipic acid hydrazide, and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.
 15. Thefiber of claim 1 crosslinked with a covalent crosslinking agent. 16-18.(canceled)
 19. A fibrous scaffold, comprising a plurality of the fibersof claim
 1. 20. (canceled)
 21. The scaffold of claim 19 furthercomprising chitosan.
 22. (canceled)
 23. The scaffold of claim 19 furthercomprising collagen.
 24. (canceled)
 25. The scaffold of claim 19 furthercomprising a biologically active amino acid molecule selected from thegroup consisting of growth factors, fibronectin, laminin, vitronectin,integrins, nucleic acid molecules, lipids, sugars, antisense molecules,anti-inflammatory agents, immunomodulatory agents, molecules thatpromote cell migration, molecules that promote cell division, moleculesthat promote cell proliferation and differentiation, molecules thatstimulate phenotypic modification of cells, molecules that promoteangiogenesis, molecules that promote vascularization, and molecules thatpromote extracellular matrix disposition.
 26. The scaffold of claim 19further comprising a signaling ligand selected from the group consistingof members of the TGF-β family, bone morphogenic proteins, fibroblastgrowth factors-1 and -2, platelet-derived growth factor-AA and -BB, andplatelet rich plasma and vascular endothelial cell-derived growthfactor. 27-28. (canceled)
 29. A method for making alginate fibers,comprising: (a) generating an electrostatic field between a firstelectrode and a second electrode; and (b) electrospinning an aqueousalginate solution comprising alginate and a hydrophilic polymer onto acollection surface located between the first electrode and the secondelectrode to provide a plurality of alginate fibers on the collectionsurface.
 30. The method of claim 29, wherein the alginate solutioncomprises from about 1 to about 8 percent by weight alginate.
 31. Themethod of claim 29, wherein the hydrophilic polymer is selected from thegroup consisting of a poly(alkylene oxide), a polyvinyl alcohol, and apolycarboxylic acid polymer.
 32. The method of claim 29, wherein thealginate solution comprises from about 1 to about 10 percent by weightpoly(alkylene oxide).
 33. (canceled)
 34. The method of claim 31, whereinthe poly(alkylene oxide) is polyethylene oxide.
 35. The method of claim31, wherein the ratio of alginate to poly(alkylene oxide) is from about70:30 to about 95:5.
 36. (canceled)
 37. The method of claim 29, whereinthe alginate solution further comprises a co-solvent.
 38. (canceled) 39.The method of claim 37, wherein the co-solvent is selected from thegroup consisting of 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile,dimethylformamide, and dimethylsulfoxide.
 40. (canceled)
 41. The methodof claim 37, wherein the co-solvent is present in the solution in anamount from about 0.5 to about 20 weight percent.
 42. (canceled)
 43. Themethod of claim 29, wherein the alginate solution further comprises asurfactant.
 44. The method of claim 43, wherein the surfactant isselected from the group consisting of glycol stearate, cocoamidesurfactants, and polyoxyethylene(10) isooctylphenyl ether.
 45. Themethod of claim 43, wherein the surfactant is present in the solution inan amount from about 0.01 to about 1.0 weight percent. 46-47. (canceled)48. The method of claim 29 further comprising treating the plurality ofalginate fibers with an ionic crosslinking agent to provide a pluralityof ionically crosslinked alginate fibers.
 49. The method of claim 48,wherein the crosslinking agent is a metal ion selected from the groupconsisting of calcium (2) ion, barium (2) ion, strontium (2), copper(2), zinc (2), magnesium (2), manganese (2), cobalt (2), lead (2), iron(3), and aluminum (3) ions.
 50. The method of claim 48 furthercomprising treating the plurality of ionically crosslinked alginatefibers with a covalent crosslinking agent to provide a plurality ofcovalently crosslinked alginate fibers.
 51. The method of claim 50,wherein the covalent crosslinking agent is a bifunctional crosslinkingagent selected from the group consisting of carbodiimides, allyl halideoxides, dialdehydes, diamines, and diisocyanates.
 52. The method ofclaim 50, wherein the covalent crosslinking agent is selected from thegroup consisting of epichlorohydrin, gluteraldehyde, hexamethylenediisocyanate, adipic acid hydrazide, and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.
 53. Themethod of claim 29 further comprising treating the plurality of alginatefibers with a covalent crosslinking agent to provide a plurality ofcovalently crosslinked alginate fibers.
 54. An implantable device,comprising the fiber of claim
 1. 55. The device of claim 54, wherein thedevice is a cell transplantation device, a drug delivery device, a wounddressing, a hemostat, a surgically implantable device for repairing adamaged cartilage in mammalian articulating joints, a fabric barrier tohyperplasia and tissue adhesion, a vascular prosthesis, a nerve graft, aspinal fusion cage, or a skin substitute.